Microdroplet manipulation method

ABSTRACT

A method of manipulating microdroplets having an average volume in the range 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of a w1  of less than 1 is provided. It is characterised by the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1. The method may be employed for example with microdroplets containing biological cells or with microdroplets containing single nucleoside phosphate such as are prepared in a droplet-based nucleic acid sequencer. The method is suitable

This invention relates to an improved method of manipulating aqueousmicrodroplets optionally containing biological cells in an immisciblecarrier fluid such as an oil. It enables the size of the microdropletsto be controlled or adjusted and any enzymatic or chemical reactionsoccurring therein to be maintained or optimised during a given period.

In our previous patent applications, for example WO2014167323,WO2015121675, WO2016012789, WO2017140839 and PCT/EP2018066574, we havedescribed methods in which biological components such as cells, enzymes,oligonucleotides and even single nucleotides are manipulated withinmicrodroplets for purposes of carrying out a range of analyses includingDNA and RNA sequencing and the detection and characterisation of cellsand viruses. In some embodiments, these methods involve translocatingmicrodroplets dispersed in an immiscible carrier fluid alongmicrofluidic pathways in an analytical device using electrowettingpropulsive forces or by directly printing of the microdroplets onto asubstrate coated with the carrier fluid. In many instances, where thevolume fraction of the microdroplets is relatively low, we have foundthat these microdroplets tend to undergo significant shrinkage over timewhich can sometimes interfere with some or all the enzymatic processesgoing on within. Also, in other instances it may be desirable todeliberately shrink or grow the size of the microdroplets in a part of adevice as a given analysis is carried out.

We have now developed a microdroplet manipulation method which overcomesthese problems. It may be used, for example, to manipulate the sizeand/or reactivity of the contents of microdroplets or to controlchemical or enzymatic reactions occurring therein. The invention is asdefined in the appended claims. According to a first aspect of theinvention, there is provided a generic method of manipulating(controlling the size and/or chemical composition of the contents of)microdroplets having an average volume in the range 0.5 femtolitres to10 nanolitres comprised of at least one biological component and a firstaqueous medium having a water activity of a_(w1) of less than 1characterised by the step of maintaining the microdroplets in awater-immiscible carrier fluid which further includes secondary dropletsof a second aqueous medium having an average volume less than 25% of theaverage volume of the microdroplets up to and including a maximum of 4femtolitres and wherein the volume ratio of carrier fluid to totalvolume of microdroplets per unit volume of the total is greater than2:1.

Without wishing to limit the scope of the invention, it is believed thatthe invention solves the problem by using a carrier fluid which containsvery small secondary droplets which can interact with the microdropletswithout adversely affecting the latter's overall characteristics or theefficacy of any detection method applied to them. When the carriermedium is an oil such a composite medium is sometimes referred to as'hydrated oil'. An important feature in this respect is that therelative water activities of the microdroplets and the secondarydroplets are controlled within certain parameters; optionally bycontinuous monitoring and/or a feed-back loop. Here, the water activityof an aqueous medium (a_(w)) is defined as the ratio of the partialvapour pressure of the aqueous medium under investigation to that ofpure water under STP conditions. Since water tends to diffuse along agradient from high to low water activity, we have found that, within theconstraints of our systems, when the water activity of the secondaqueous medium (a_(w2)) is higher than that of the first aqueous medium(a_(w1)) the net effect is for the microdroplets to undergo expansionuntil the water activities of the two components equalise. Conversely,when the water activity of the second aqueous medium is higher than thatof the first aqueous medium the microdroplets will tend to shrink untilthese water activities equalise. In one useful embodiment, the wateractivities of the first and second aqueous media may be the same orsubstantially the same so that any tendency for the microdroplets toshrink or expand is continuously counteracted. Thus, the sizes of themicrodroplets may always be preserved. We have also found that, by thesemeans, these secondary droplets can be used to assist in preserving oreven enhancing any enzymatic or chemical reactions occurring in themicrodroplets; for example, by using the secondary droplets to feedcell-growth components to the microdroplets at one or more points in anydevice employing the method. The first and second aqueous medium mayhave compositions which in one embodiment are identical.

Thus, in one embodiment of the invention, the water activity of thefirst and the second aqueous media are independently in the range from0.9 to 1. In another embodiment, the water activity of the first aqueousmedium is from 0.9 to less than 1. In yet another embodiment, the ratioof the water activities of the first and second aqueous media(a_(w1):a_(w2)) is in the range 0.9:1 to 1:0.9.

One convenient way to perform the manipulation is using first and secondmedia which are buffers; and, if required, by varying the relativecompositions of the two. For example, in one application the ionicstrength of the first aqueous medium is in the range from to 1 to 5 thatof the second aqueous medium; preferably from 3 to 5 times. In another,the ionic strength of the second aqueous medium is in the range from to1 to 5 times that of the first aqueous medium; preferably from 3 to 5times. In yet another application, the ionic strengths are the same orsubstantially the same with the ratio of ionic strengths being in therange from 3:1 to 1:3. In one particularly useful embodiment, either orboth first and second aqueous media may include glycerol as a component;for example, at differing concentrations. In another, the pHs of thefirst and second aqueous media are the same or similar and within therange 6.5 to 8.

As regards the secondary droplets, these have a much smaller averagevolume than the average value for the microdroplets and at the limit maybe comprised of femto-sized droplets or micelles of the second aqueousmedium emulsified within the carrier fluid and stabilised by a sheath ofcompatible surfactant molecules; for example, a non-ionic surfactant. Inone embodiment, the size of these secondary droplets is less than 10%,preferably less than 5% of the volume of the microdroplets employed. Inanother, the average volume of the secondary droplets lies within therange 10 to 1% of the average volume of the microdroplets. Suitably thesecondary droplets form part of a stable emulsion in the carrier fluidwhich in one embodiment is an immiscible oil. Suitably, the carrierfluid is selected from a mineral oil, a silicone oil or a fluorocarbonoil. The oil may also contain additional surfactants and stabilisers ifrequired. Suitably the volume ratio of carrier fluid to total volume ofthe microdroplets is greater than 3:1; preferably 5:1 or greater.

The method of the invention is useful for several applications wherebiological cells are being analysed. One example is where a culture ofimmortalised mammalian cells is being caused to proliferate inside themicrodroplets for the purpose of screening individual clonal copies ofthe cells for desirable characteristics such as protein expression orparticular genetic traits. Thus in a second aspect of the invention,there is in one embodiment provided a method of causing the cellularproliferation of one or more cell types contained within a microdroplethaving an average volume in the range 4 femtolitres to 10 nanolitres andcomprised of an aqueous buffer comprising the steps of incubating thecell(s) inside the droplets in suitable environmental conditions andthereafter detecting the number of cells inside each droplet,characterised in that the microdroplets are suspended in an immisciblecarrier fluid further comprising secondary droplets having an averagevolume less than 25% of the average volume of the microdroplets up toand including a maximum of 4 femtolitres and wherein the volume ratio ofcarrier fluid to total volume of microdroplets per unit volume of thetotal is greater than 2:1.

In another embodiment, there is also provided a method of detecting oneor more phenotypic traits, genetic traits or protein expression profilesof a cell under consideration, that cell being contained within amicrodroplet having an average volume in the range 4 femtolitres to 10nanolitres and comprised of an aqueous growth media comprising the stepsof labelling a target derived from the cell(s)with a fluorescent probeand thereafter detecting an output from the probe characterised in thatthe cell-containing microdroplets are suspended in an immiscible carrierfluid further comprising secondary droplets having an average volumeless than 25% of the average volume of the microdroplets up to andincluding a maximum of 4 femtolitres and wherein the volume ratio ofcarrier fluid to total volume of microdroplets per unit volume of thetotal is greater than 2:1. Fluorescent probe molecules suitable for thispurpose are well known and include fluorescently labelled antibodies,FRET reporter probes and enzyme-labelled antigens which are degraded inthe presence of a target protein.

In another embodiment, there is provided a method of analysing anoligonucleotide derived from a biological cell contained within amicrodroplet having an average volume in the range 4 femtolitres to 10nanolitres and further comprised of an aqueous buffer comprising thesteps of labelling the oligonucleotide with a fluorescent hybridisationprobe and thereafter detecting the corresponding fluorescencecharacterised in that the microdroplets are suspended in an immisciblecarrier fluid further comprising secondary droplets having an averagevolume less than 25% of the average volume of the microdroplets up toand including a maximum of 4 femtolitres and wherein the volume ratio ofcarrier fluid to total volume of microdroplets per unit volume of thetotal is greater than 2:1.

Fluorescent hybridisation probes which can be used for this purpose arewell-known in the art and include molecular beacons, TaqMan® probes,Scorpion® probes and LNA® probes. Methods for detecting the fluorescencearising in all these embodiments are well-known to one of ordinary skillin the art; for example, those methods employing a source of incidentelectromagnetic radiation (laser, LED and the like) and a correspondingphotodetector for detecting fluorescence photons and outputting adata-stream which can be analysed using microprocessor algorithms.

Thus, the target in these methods may be the cell(s) themselves, one ormore oligonucleotides derived therefrom or a product such a proteinwhich is expressed by the cell(s) when cultured within the microdropletitself. Such oligonucleotides may be generated from the cell(s) bylysis.

The method of the invention may also be suitably employed in connectionwith biological components which are non-cellular or cell-free althoughin one embodiment it may be used to manipulate nucleic acids orcomponents thereof which have been previously derived from biologicalcells. Thus in a third aspect of the invention there is provided thereis provided a method of manipulating the size and/or reactivity of thecontents of microdroplets having an average volume in the range 0.5femtolitres to 10 nanolitres; the microdroplets being comprised of atleast one biological component and a first aqueous medium free ofbiological cells having a water activity of a_(w1) of less than 1characterised by the step of maintaining the microdroplets in awater-immiscible carrier fluid which further includes secondary dropletscomprised of a second aqueous and having an average volume less than 25%of the average volume of the microdroplets up to and including a maximumof 0.5 femtolitres and wherein the volume ratio of carrier fluid tototal volume of microdroplets per unit volume of the total is greaterthan 2:1.

The method of the third aspect of the invention is useful for a numberof applications where the biological component is a single nucleotide;for example, a single nucleoside triphosphate or single nucleosidemonophosphate. For example, the method may be advantageously used withone of the sequencing methods we have previously described including butnot limited to those described EP3013987 or in the other above-mentionedpatent applications to which the reader is directed. Thus, in a thirdaspect, there is provided a method of sequencing comprising the steps ofprogressively digesting by pyrophosphorolysis a nucleic acid analyteinto an ordered stream of nucleoside triphosphate molecules andgenerating therefrom a corresponding ordered stream of microdropletshaving an average volume in the range 0.5 femtolitres to 10 nanolitresand each comprised of one of the nucleoside triphosphate molecules andaqueous buffer; reacting each nucleoside triphosphate molecule withineach microdroplet with a nucleobase-specific fluorescent probe andthereafter detecting the corresponding fluorescence associated with eachmicrodroplet thereby identifying the nucleobase characterised in thatthe microdroplets are suspended in an immiscible carrier fluid furthercomprising secondary droplets having an average volume less than 25% ofthe average volume of the microdroplets up to and including a maximum of0.5 femtolitres and wherein the volume ratio of carrier fluid to totalvolume of microdroplets per unit volume of the total is greater than2:1.

Fluorescent probes suitable for use in this application have beendescribe by us in our previous patent applications; for example,WO2016012789 and subsequently published applications to which the readeris directed. These probes are characterised by (a) being non-fluorescingin their unused state and (b) being capable of undergoing exonucleolysisonce used to produced fluorophores in a detectable state attached tosingle nucleoside monophosphates. The fluorescence arising may bedetected and analysed as described above.

In all these additional aspects of the invention it is preferred thatthe ratio of the water activities of the first and second aqueous mediaassociated with respectively the microdroplets and the secondarydroplets is in the range 0.9:1 to 1:0.9; preferably 0.95:1 to 1:0.95 andfor example 1:1.

The advantageous effect of hydrating the carrier phase as describedabove is now illustrated by the following Examples.

Example 1 (Cell Growth)

Continuous oil phase material is prepared by mixing 99 parts of aHydrofluoroether continuous phase with 1 part of a fluorinatedsurfactant. A growth-media-treated carrier phase is prepared by mixingan aliquot of RPMI 1640 media (Thermo Fisher Scientific, UK) with anequal volume of the oil/surfactant mixture and agitating the mixture for24 hours at 37° C. to form a polydisperse emulsion. This emulsion isthen left to stand until it spontaneously fractionates to form an upperphase comprising large droplets and undispersed plugs of aqueous growthmedia, and a lower phase containing only the smallest vesicles of growthmedia suspended in the oil phase which is additionally now saturatedwith dissolved aqueous media. This lower phase is removed from thevessel using a pipette and retained for later use.

Jurkat E6-1 T-cell lymphoma cells (ATCC, Virginia, USA) are suspended inRPMI media at a concentration of 8E6 cells/ml. This media and cells arethen flowed through an emulsifying apparatus to form droplets of 50 umdiameter, with cells dispersed throughout the droplets. The outercarrier phase for the emulsion is a hydrofluoroether oil mixed with 1%of a suitable surfactant to stabilise the droplets in solution. Theemulsion thus formed spontaneously fractionates to form a layer ofdensely packed monodisperse aqueous droplets floating at the top of acolumn of continuous oil/surfactant mixture. This emulsion is thenevenly dispersed by gentle mixing and divided in to three aliquotscontaining droplets and the carrier phase.

One aliquot (the initial reference) is immediately transferred in to ahaemocytometer flow cell and the droplets therein are inspected using a20x magnification optical microscope. The cell occupancy of each dropletis recorded by counting the number of distinct cells in each droplet.Empty droplets are disregarded.

The second aliquot is allowed to fractionate once more, and the lowercarrier phase removed using a pipette. An equivalent volume of theearlier treated carrier phase is introduced to the sample to replace theremoved untreated carrier phase. The third aliquot is left unaltered.Both the second and third aliquots are then transferred in to partiallysealed vessels which permit gas permeation between the vessel and itssurroundings. Both vessels are placed in to an environment-controlledCO2 incubator set to contain 5% CO2/air mixture, 95% humidity and 37° C.set temperature. The aliquots are incubated for 24 hours

These aliquots are then removed from the incubator and introduced to ahaemocytometer for inspection and analysis in the same way as thereference aliquot. The change in the cell population-distribution afterthe incubation (characteristic of cell proliferation) can then becompared between the different oil treatments.

FIG. 1 compares the results obtained after 24 hours culture relative tobaseline measurement at zero and 24 hours with no hydration of the oil.Cell growth is expressed here as a fraction of the droplets containingmore than one cell. It will be seen that, relative to the baseline cellgrowth, an improvement occurs when the oil is hydrated with the cellculture medium.

Example 2 (Reactivity)

A continuous hydrated oil phase is prepared by mixing 99 parts of alight mineral oil with 1 part of a pegylated surfactant by weight. Theoil is placed on rotator overnight to fully mix oil and surfactant.Hydrated oil is prepared by mixing 5 parts of the oil with 3 parts ofthe aqueous hydrating phase, consisting of either the same saline bufferused in the disperse emulsion phase or just water. The mixture isrotated overnight at 50C and then for 60 min at 70C. The emulsion isleft to stand for 15 min. The upper portion of the emulsion isaliquoted, and the aliquots are then centrifugated to adjust thehydration level of the oil, with longer centrifugation times leading tolower hydration levels. The hydration level is measured using a KarlFisher titrator. Once the correct hydration level is achieved, usually500-1000 ppm, the supernatant of the aliquots is pipetted into new tubeswhich are frozen until used.

A polydisperse emulsion of droplets is produced by mixing 8 parts of theoil (either hydrated or not hydrated) with 1 part of the disperseaqueous phase by volume followed by mixing on a vortex mixer for 5 minand centrifugation for 1 min at 400 RPM. The upper half of the mixtureis pipetted into a new tube which is centrifuged for 5s. The emulsion ispipetted from the bottom of the tube for further use.

For measurements of the enzymatic activity, the disperse aqueous phasedescribed above consists of the single nucleotide detection chemistry aspreviously described and exemplified in EP3013987.

For measurement of the fluorescent intensity the emulsion is sandwichedbetween two transparent substrates separated by spacers corresponding tothe average emulsion droplet size. The fluorescent signal emitted fromeach emulsion droplet, upon excitation with light of an appropriatewavelength range, is measured together with the diameter of dropletwhich is collected from a brightfield image of the emulsion.

The data presented as a histogram in FIG. 2 shows the averagefluorescent intensity of 6um droplets, which have been incubated in oilwith no hydration ('Dry oil'), in oil hydrated with water ('Water only')or oil hydrated with three times the buffer concentration of thedroplets ('3x buffer'). Droplets incubated in oil with no hydration showvery low intensity above the background which for these samples isapproximately 1000 counts. Those incubated in oil hydrated with watershow an increased intensity compared to droplets incubated in oil withno hydration. Incubation of droplets in oil hydrated with three timesthe buffer concentration shows a further increase in average intensity.This demonstrates that both oil hydration can be used to maintainenzymatic reactivity in these droplets.

Example 3 (Droplet Size Effect)

Microdroplets are deposited on a substrate immersed in a continuous oilphase as for example previously described in EP3008207 to which thereader is directed.

The continuous hydrated oil phase is prepared by mixing 99 parts ofparaffin oil with 1 part of a pegylated surfactant by weight. The oil isplaced on rotator overnight to fully mix oil and surfactant. Hydratedoil is prepared by mixing 5 parts of the oil with 3 parts of the aqueoushydrating phase, consisting of water with or without 4% glycerol. Themixture is rotated overnight at 50C and then for 60 min at 70C. Theemulsion is left to stand for 15 min. The upper portion of the emulsionis aliquoted, and the aliquots are then centrifugated to adjust thehydration level of the oil, with longer centrifugation times leading tolower hydration levels. The hydration level is measured using a KarlFisher titrator. Once the correct hydration level is achieved, usually500-1000 ppm, the supernatant of the aliquots is pipetted into new tubeswhich are frozen until used.

The disperse aqueous phase consists of water with or without 4%glycerol. The deposited droplets are subjected to an incubation cycle at70C for 115 min. The emulsion droplet diameters are then measured from abrightfield microscope image and compared to measured diameters prior tothe incubation cycle to infer droplet shrinkage or growth.

The data presented below shows the average volume change of dropletsupon a high temperature incubation step as a function of percentage ofglycerol in the oil hydration and droplets respectively. In thereference sample, if glycerol is not present in either the oil hydrationnor in the droplets, the droplets shrink on average. If the dropletscontain glycerol whereas the oil hydration does not, the droplets growrelative to the reference because the addition of glycerol to thedroplets causes the water activity in the oil to be higher than thewater activity in the droplets. The reverse happens when glycerol isadded to the oil hydration but not the droplets. Droplets shrinkrelative to the reference due to a higher water activity in the dropletscompared to the oil. This shows that the specific content of thedroplets and the oil hydration can be used to control droplet shrinkageand growth.

% glycerol in oil hydration % glycerol in droplets Droplet volume change0 0 -30% (shrink) 0 4 +44% (grow) 4 0 -51% (shrink)

1-17. (canceled)
 18. A method of controlling the size of microdroplets and/or maintaining or optimising enzymatic or chemical reactions occurring therein, said microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres comprised of at least one biological component and a first aqueous medium having a water activity of a_(w1) of less than 1 comprising the step of maintaining the microdroplets in a water-immiscible carrier fluid which further includes secondary droplets comprised of a second aqueous medium having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 19. The method of claim 18 for manipulating the size and/or chemical or enzymatic reactivity of the contents of microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres, the microdroplets being comprised of at least one biological component and a first aqueous medium free of biological cells having a water activity of a_(w1) of less than 1 wherein the step of maintaining the microdroplets in a water-immiscible carrier fluid further includes secondary droplets comprised of a second aqueous medium and having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 0.5 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 20. The method of claim 18 for controlling chemical or enzymatic reactivity and/or microdroplet size in microdroplets having an average volume in the range from 4 femtolitres to 10 nanolitres, the microdroplets being comprised of at least one biological cell and a first aqueous medium having a water activity of a_(w1) of less than 1 wherein the step of maintaining the microdroplets in a water-immiscible carrier fluid further includes secondary droplets comprised of a second aqueous medium and having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 21. The method of claim 18, wherein the secondary droplets have a water activity a_(w2) which is greater than a_(w1).
 22. The method of claim 18, wherein the secondary droplets have a water activity a_(w2) which is less than a_(w1).
 23. The method of claim 18, wherein water activities a_(w1) and a_(w2) are the same.
 24. The method of claim 18, wherein a_(w1) and a_(w2) are independently in the range 0.9 to
 1. 25. The method of claim 21, wherein the ionic strength of the second aqueous medium is in the range from 1 to 5 times that of the first aqueous medium.
 26. The method of claim 22, wherein the ionic strength of the first aqueous medium is in the range from 1 to 5 times that of the second aqueous medium.
 27. The method of claim 18, wherein the average volume of the secondary droplets is less than 10% of the average volume of the microdroplets.
 28. The method of claim 18, wherein at least one of the first and second aqueous media further comprise glycerol.
 29. The method of claim 18, wherein the biological component is selected from a single nucleoside triphosphate derived from a target nucleic acid, an oligonucleotide derived from the DNA or RNA of a cell, an enzyme or a cell.
 30. The method of claim 29, wherein the first and/or second aqueous media are buffers.
 31. A method of causing the cellular proliferation of one or more cell types contained within a microdroplet having an average volume in the range 4 femtolitres to 10 nanolitres and comprised of an aqueous buffer comprising the steps of incubating the cell(s) inside the droplets in suitable environmental conditions and thereafter detecting the number of cells inside each droplet, wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 32. A method of analysing or detecting one or more phenotypic traits, genetic traits or protein expression profiles of a cell under consideration, that cell being contained within a microdroplet having an average volume in the range 4 femtolitres to 10 nanolitres and comprised of an aqueous buffer comprising the steps of labelling a target derived from the cell(s) wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 4 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 33. A method of sequencing comprising the steps of progressively digesting by pyrophosphorolysis a nucleic acid analyte into an ordered stream of nucleoside triphosphate molecules and generating therefrom a corresponding ordered stream of microdroplets having an average volume in the range from 0.5 femtolitres to 10 nanolitres and each comprised of one of the nucleoside triphosphate molecules and aqueous buffer; reacting each nucleoside triphosphate molecule within each microdroplet with a nucleobase-specific fluorescent probe and thereafter detecting the corresponding fluorescence associated with each microdroplet thereby identifying the nucleobase wherein the microdroplets are suspended in an immiscible carrier fluid further comprising secondary droplets having an average volume less than 25% of the average volume of the microdroplets up to and including a maximum of 0.5 femtolitres and wherein the volume ratio of carrier fluid to total volume of microdroplets per unit volume of the total is greater than 2:1.
 34. The method of claim 31, wherein the ratio of the water activities of the microdroplets and the secondary droplets is in the range 0.9:1 to 1:0.9; preferably 0.95:1 to 1:0.95. 